This invention relates to a scanning confocal optical microscope system.
The confocal scanning optical microscope is now widely used. In its essentials it consists of a means for focussing a beam of light to a small spot on a specimen and means for collecting the emitted or reflected emission from that spot in order to build up an image by systematic scanning of the spot over a specimen. A defining feature of the confocal instrument is the presence of a beam-limiting aperture in front of the detector, which serves to limit detection of emitted light to that emerging from the immediate vicinity of the focus of illumination. White, in U.S. Pat. No. 5 032 720, taught the use of a variable iris as such an aperture, allowing a compromise to be sought between best optical depth discrimination, which is obtained with the aperture maximally closed, and high signal strength, which is obtained with it open. An analysis of this advantage has been published by Sandison et al, pp 39-51 in Handbook of Biological Confocal Microscopy, IInd Edn., Plenum Press, New York and London, 1995. White also taught the division of the emitted light in a confocal microscope into more than one beam according to wavelength, by means of chromatic reflectors. This principle has proved to have many applications, chiefly in the imaging of a plurality of fluorescent stains simultaneously present in the same specimen.
In a widely-used commercial form of White""s instrument, there are two or three such beams, each passing light to a separate variable iris, i.e. there are two or three confocal beam-limiting apertures. The value of having more than one aperture is that the diameter may be set differently in each. This is of value because the above-mentioned compromise may be sought according to the brightness of each individual stain. Also, the theoretical optimum width of the aperture scales with wavelength, so a single diameter can never be precisely optimum for all wavelengths (van der Voort, H. T. M. and Brakenhoff, G. J. (1990), J. Microscopy 158, pp 43-54).
In White""s microscope system, the separation of beams is achieved by the use of chromatic reflectors and further colour separation is achieved by means of barrier filters. Since it is accepted to be desirable to be able to distinguish colours, the use of a spectrometer in the emission path of a confocal microscope is an obvious development. Brakenhoff, in a diagram published on page 189 of Confocal Microscopy, edited by T. Wilson, Academic Press 1990, showed how a spectrometer could be used. FIG. 1 of the accompanying drawings is redrawn from Brakenhoff""s figure and serves to clarify the placement of components in a confocal microscope.
In FIG. 1, light from a laser 11 is passed through a lens 12 and a first aperture, consisting of a pinhole 13, from which the light emerges as an expanding beam which is rendered parallel by lens 14. From lens 14 the light is reflected by chromatic reflector 15 so that it passes into a microscope objective lens 16 and is brought to a focus, normally as a diffraction-limited spot on a specimen at 17. Some of the light emitted from the specimen passes into lens 16 and the chromatic performance of the reflector 15 is chosen such that the emitted light is transmitted by this reflector 15 rather than reflected. The transmitted beam passes through a barrier filter 18, which absorbs unwanted laser light, and is focussed by the lens 19 on a confocal aperture at 20. It is essential for the proper functioning of a confocal microscope that this aperture lies in an optically conjugate position to the focus on the specimen; in other words the specimen is focussed on this aperture. In conventional optical terminology, the confocal aperture is an image plane stop.
FIG. 1 shows how the light which has passed through the confocal aperture at 20 is passed, in Brakenhoff""s scheme, through a spectral dispersing means, such as a monochromator 21, and the outgoing light is finally passed to a unitary detector such as a photomultiplier tube 22. The photocurrent in the photomultiplier tube 22 is used as a measure of the intensity of the light in the range of wavelengths selected by the monochromator 21 and allows the construction of an image in computer memory if the spot of light is scanned systematically over the specimen.
In order to be able to record images from the spectrometer (photomultiplier tube 22) at more than one wavelength simultaneously, it is a possible development of the system proposed by Brakenhoff that the spectrometer should be of the multichannel type. This was proposed explicitly by Engelhardt in PCT Application WO 95/07447. This combination of the known art of spectrometry with known apparatus for confocal microscopy works well and has the advantage of being more flexible than the fixed-reflector design taught by White. It is, however, inferior to White""s design in that a plurality of confocal apertures, each for a different wavelength range, cannot be used. Since all the detected light is passed through a single confocal aperture the previously mentioned advantages of multiple apertures are lost.
The present invention aims to overcome this difficulty, allowing the use of multiple confocal apertures in conjunction with multichannel spectral detection. It effectively consists of a form of imaging spectrometer, which is simple in construction and easily integrated into a scanning confocal optical microscope system.
According to one aspect of the invention, there is provided a scanning confocal microscope system which is confocal in operation, the system including a dispersive optical means which produces from the same restricted region of a specimen a plurality of separated optical images of differing wavelength ranges, and a beam-limiting aperture for each said image, all said apertures being located at foci which are conjugate with each other. All the apertures are located in image planes which are conjugate with the plane of focus in the specimen, and the central points of the apertures are conjugate with the point of illumination in the specimen. The above-mentioned beam-limiting image plane apertures function as confocal apertures.
Preferably, the restricted region or area of the specimen is imaged at a primary image plane and the dispersive optical means, in conjunction with focussing means, produces said images of differing wavelength ranges in secondary image planes conjugate with the primary image plane.
The system preferably includes a plurality of detectors receiving light through the respective beam-limiting apertures.
According to another aspect of the invention, there is provided a scanning confocal optical microscope system comprising a scanning confocal optical microscope which produces a beam of light forming an image of a restricted region of a specimen in a primary image plane, a dispersive optical means which receives the beam from the said primary image plane and produces, in secondary image planes each conjugate with the primary image plane, a plurality of separated secondary optical images of the same region of the specimen, said secondary images respectively comprising light of differing ranges of wavelengths, a beam-limiting aperture in each secondary image plane and a plurality of detectors receiving light through the respective beam-limiting apertures.
Preferably, the beam-limiting area of at least one of the beam-limiting apertures is adjustable or variable. This may be achieved by exchange of a beam-limiting aperture of one size by an aperture of a different size, or by making the beam-imiting area adjustable in size, e.g. in width or diameter. For example, at least one beam-limiting aperture may comprise a variable iris diaphragm, a system of movable jaws, or an adjustable slit.
The dispersive optical means may comprise one or more optical prisms or a diffraction grating or gratings, but an equivalent device or devices may alternatively be employed.
Conveniently, a focusing means such as a lens reproduces the image in the primary image plane in the aperture plane of a second focusing means, e.g. a lens, whereby the aperture or exit pupil of the first focusing means is imaged at a location beyond the dispersive optical means (i.e. the dispersive means is located between the second focusing means and said location), whereby a spread of spectrally differing images of the aperture of the first focusing means is generated.
Conveniently, an optical beam-separating means, such as a reflector or reflectors, may be provided, whereby to direct the light from different points of the spread of spectrally differing images to the beam-limiting apertures, at which are formed a spatially spread series of images of the primary image plane, each confined to a wavelength range different to the wavelength range at the other beam-limiting apertures. The beam-separating means may be adjustable to enable variation of the wavelength range of the image at each beam-limiting aperture. As well as the beam-separating means, a further focusing means may be provided to generate the relayed images at the said confocal apertures.
A further reflector or reflectors or other further optical beam-separating means may serve to spatially spread the relayed images.
The wavelength compositions of the spectrally spread images may be variable, as by adjustment of the beam-separating means, e.g. the reflector or reflectors, and/or by the positioning of wavelength-sensitive absorbing screens or filters into the path or paths of the spectrally spread images.